Structural features of the vitamin K-dependent blood coagulation proteins will be examined by immunochemical and spectroscopic methods to gain insight into the special functional features of these proteins. In order to identify the role of individual gamma-carboxyglutamic acid (Gla) residues in prothrombin, partially decarboxylated prothrombin will be prepared by heat decarboxylation of prothrombin. These species will be purified and extensively characterized with regard to metal binding properties, Gla content, sequence, and conformation. In particular, conformation-specific antibodies specific for prothrombin or for des-gamma-carboxy prothrombin will be used to assess tertiary structure. Gla-containing peptides will be synthesized as model compounds, and then metal binding properties examined by 13C NMR relaxation studies. In a separate study, anti-human abnormal prothrombin-specific antibodies will be prepared and used to measure abnormal prothrombin in plasma using an RIA. Assay of abnormal prothrombin in plasma obtained from patients with vitamin K deficiency, DIC, liver disease, hemorrhagic disease of newborn, or those given warfarin will be used to evaluate hepatic carboxylase activity and the adequacy of vitamin K levels in these conditions. Lastly, studies using conformationally-specific antibodies as probes of the structure of prothrombin, abnormal prothrombin, and Factor X will continue. Fragments of these proteins will be prepared proteolytically from the native protein or by solid phase peptide synthesis. These fragments will be used to purify antibody subpopulations by sequential immunoabsorption using affinity chromatography. These antibody subpopulations will be used to determine domains of prothrombin whose structure is metal-stabilized, domains of Factor X that are conformationally-altered during zymogen activation and regions of abnormal prothrombin which are immunochemically distinct from prothrombin. As a new strategy to the use of antibodies as probes of protein structure, hybridona cell lines will be raised and their monoclonal antibodies evaluated as reagents for structural studies of the vitamin K-dependent proteins. These studies should further our understanding of the role of gamma-carboxyglutamic acid and the vitamin K-dependent proteins in blood coagulation.